Molecular analysis of the UV protection and mutation genes carried by the I incompatibility group plasmid TP110.

The abortive infection of bacteriophage T7 in Shigella sonnei D2 371-48 is characterized by a premature inhibition of phage DNA replication and nucleolytic breakdown of all phage DNA. Mutations in T7 gene 10 which are recessive to the presence of the wild-type allele can alleviate the restriction of phage growth. Phage T3 productively infects S. sonnei D2 371-48, as does a T7-T3 hybrid phage that contains, in particular, a gene 10 of T7 origin. It is the presence of T3 DNA ligase that allows phage growth on S. sonnei D2 371-48, and this enzyme can also rescue wild-type T7 from the abortive infection. T7+ is therefore functionally ligase deficient during the infection of S. sonnei D2 371-48; this deficiency is a result of the expression of the phage capsid protein, but it is independent of the assembly of the protein into a procapsid or other morphogenetic structure.     

Location of the ss--mutation of bacteriophage T7 in genes 10, the structural gene for the major capsid protein.

T7+ phage are unable to plate on a strain of Shigella sonnei D2 371-48. Spontaneous phage mutants arise (ss--mutants) that are able to plate on this strain of Shigella. We have shown by complementation studies and genetic crosses that the ss--mutation maps in gene 10, the structural gene for the major protein of the capsid. This finding implies that the gene 10 protein may interact with a host protein during phage development and that the abortive infection of T7 observed in S. sonnei D2 371-48 with T7+ phage may be a defect in head morphogenesis. Our studies also reveal that various T7 strains commonly contain deletions in nonessential regions. T7 ss--mutants selected after growth of T7+ on Shigella D2 371-48 often acquire a deletion in the 0.7 gene that is not necessary for the ss--phenotype. Finally, we have found a new nonessential region of the T7 chromosome that is located between 33 and 35.5% of the T7 genome length.     

A single-base change in gene 10 of bacteriophage T7 permits growth on Shigella sonnei.

Bacteriophage T7 can extend its host range to include Shigella sonnei D2 371-48 by a mutation called ss found in the T7 major capsid protein, the gene 10 product. We show that a single A-to-C transversion at position 23150 in the T7 genome is responsible for the T7 ss mutant phenotype that allows the phage to avoid DNA degradation and undergo productive infection. The ss mutation causes an amino acid substitution of proline for glutamine at position 61 of the 344-amino-acid T7 major capsid protein.     

Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.     

Amber Mutants of Bacteriophages T3 and T7 Defective in Phage-directed Deoxyribonucleic Acid Synthesis

Amber mutants of the related phages T3 and T7 were isolated and tested for their ability to restore-as the wild type does-thymidine incorporation in ultraviolet (UV)-irradiated, UV-sensitive, nonpermissive host bacteria (Escherichia coli B(s-1)). Most amber mutants had this ability. However, in both T3 and T7, mutants unable to promote thymidine incorporation under these conditions were found and classified into two well-defined complementation groups: T3DO-A and T3DO-B, T7DO-A and T7DO-B. Infection of B(s-1) cells with representatives of groups DO-A had the following characteristics: (i) phage-directed uridine uptake in UV-irradiated cells was reduced to less than 20% of normal; (ii) breakdown of host deoxyribonucleic acid (DNA) was delayed and incomplete; (iii) no serum-blocking antigens appeared; (iv) no cell lysis occurred; (v) the ability to exclude the heterologous wild type was impaired. Amber mutants of the DO-B groups, infecting B(s-1), were able to: (i) promote an efficient phage-directed uridine uptake in UV-irradiated cells; (ii) bring about rapid breakdown of host DNA; (iii) synthesize serum-blocking antigens; (iv) lyse the host cells, generally after the normal latent period; (v) exclude efficiently the heterologous wild type. Although physiological similarities between the respective DO-A mutants or DO-B mutants of T3 and T7 were evident, no physiological cross-complementation occurred, and genetic crosses gave no evidence of genetic homologies between groups of T3 and T7.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Host-controlled Restriction of T-even Bacteriophages: Relation of Four Bacterial Deoxyribonucleases to Restriction

Escherichia coli strains B and K-12, which restrict growth of nonglucosylated T- even phage (T(*) phage), and nonrestricting strains (Shigella sonnei and mutants of E. coli B) were tested for levels of endonuclease I and exonucleases I, II, and III, by means of in vitro assyas. Cell-free extracts freed from deoxyribonucleic acid (DNA) were examined with three substrates: E. coli DNA, T(*)2 DNA, and T2 DNA. Both restricting and nonrestricting strains had comparable levels of the four nuclease activities and had similar patterns of preference for the three substrates. In addition, mutants of E. coli B and K-12 that lack endonuclease I were as effective as their respective wild types in restricting T(*) phage.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Early Abortive Lysis by Phage BF23 in Escherichia coli K-12 Carrying the Colicin Ib Factor

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.     

Origin of Polysaccharide Depolymerase Associated with Bacteriophage Infection

Analyses, by construction of phage growth curves, indicated that the polysaccharide depolymerase was synthesized by Pseudomonas aeruginosa strains B and BI after infection with phage 2. The kinetics of biosynthesis of the depolymerase were found to parallel closely the rate of formation of phage-directed virions, and alterations in the experimental conditions of infection were reflected by alterations in the production of enzyme. Infection with other Pseudomonas phages, 84 and 1197, did not result in the synthesis of depolymerase. The enzyme was not detectable in uninfected cultures, and no evidence was obtained for the existence of inhibitors or activators of enzyme activity in extracts of uninfected or infected cells. The results of experiments employing chloramphenicol or an auxotorphic mutant (BI arg(-)) suggested that protein synthesis de novo was essential for production of the enzyme. Various mutants of phage 2 (pdp(1), pdp(2)), which alter the synthesis of the polysaccharide depolymerase, have been isolated. These experimental results strongly support the role of the phage genome in the synthesis of this enzyme.     

Complete genomic sequence of the lytic bacteriophage phiYeO3-12 of Yersinia enterocolitica serotype O:3

phiYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted phiYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, phiYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D(2)371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported.     

Bacteriocinlike killing action of a temperate bacteriophage phiBA1 of Bacillus aneurinolyticus.

A new temperate phage, phiBA1, was isolated from Bacillus aneurinolyticus, phiBA1 had an icosahedral head with a diameter of about 70 nm and a tail about 20 nm long and contained a circularly permuted, linear duplex DNA of about 38 x 106 daltons. This phage showed two activities: bacteriocin-like killing activity against five strains of B. aneurinolyticus and normal temperate phage activity against three other strains. phiBA1 killed sensitive cells by a single-hit process. After adsorption of phiBA1 to cells sensitive to killing, the content of intracellular ATP increased for the first 5 min and then gradually decreased. Phage DNA injected into the cell immediately after infection was degraded rapidly. Killing was also caused by heavily UV-irradiated phiBA1. Killing-resistant mutants showed normal adsorption of phiBA1 and normal injection of the DNA with its instantaneous restriction. Our results indicate that the killing action of phiBA1 is different from the phenomenon of abortive infection and suggest that the killing might be caused by a proteinaceous component of phiBA1.     

Excision of Thymine Dimers In Vitro by Extracts of Bacteriophage-Infected Escherichia coli

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from UV-irradiated DNA previously nicked with T4 UV endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 UV-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. A similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Growth of Coliphage T7 in Salmonella typhimurium

A mutant of Salmonella typhimurium was found to be sensitive to killing by coliphage T7 because of an alteration in its surface properties. However, the infections were abortive and studies with (32)P-labeled T7 grown in Escherichia coli B (T7.B) indicated that the phage DNA was restricted by S. typhimurium. When a mutant T7 which survived the restriction and produced plaques on Salmonella (T7.S) was passed through one cycle of growth in E. coli B, its ability to grow in Salmonella was lost, indicating that host-controlled restriction and modification are operative in this system. Restrictionless S. typhimurium mutants were isolated that permit the growth of not only T7.S but also T7.B and coliphage T3. The physiology of T7 production in the restrictionless host is nearly identical to that in Escherichia coli.     

das Mutation in bacteriophage T4D does not suppress an amber mutation in T4 gene 59.

Mutations termed das were isolated originally (Hercules and Wiberg, 1971) as partial suppressors of mutants in phage T4 genes 46 and 47. Since mutants in genes 46, 47, and 59 exhibit both an early arrest of phage DNA synthesis and the loss of this arrest in the presence of chloramphenicol or of mutations of T4 genes 33 and 55, we asked whether a das mutation can also suppress a gene 59 mutant. We find that it cannot--either at the level of phage production or DNA synthesis.     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

Role of gene 2 in bacteriophage T7 DNA synthesis.

Studies have been carried out to elucidate the in vivo function of gene 2 in T7 DNA synthesis. In gene 2-infected cells the rate of incorporation of (3-H)thymidine into acid-insoluble material is about 60% that of cells infected with T7 wild type. Gene 2 mutants do not however produce viable phage after infection of the nonpermissive host. In T7 wild type-infected cells, a major portion of the newly alkaline sucrose gradients. The concatemers serve as precursors for the formation of mature T7 DNA as demonstrated in pulse-chase experiments. In similar studies carried out with gene 2-infected cells, concatemers are not detected when the intracellular DNA is analyzed at several different times during the infection process. The DNA made during a gene 2 infection is present as duplex structures with a sedimentation rate close to mature T7 DNA.